In previous studies, we found that the immunoglobulin kappa gene 3' enhancer is controlled by a variety of transcription factors (PU.1, Pip, E47, c-jun, c-fos, ATF1, CREM, Sp1, BSAP, and YY1). These factors can bind the 3' enhancer in vitro and can control its activity in transient expression assays. How these factors control the developmental activity of the enhancer in vivo is unknown. Although in vivo footprinting studies have shown differences in enhancer occupancy at distinct developmental stages, the specific proteins responsible for these differences are unknown. We will use chromatin immunoprecipitation (CHIP) and DNA pull down assays to identify the specific proteins that bind to the 3' enhancer at defined stages of B cell development. We will also determine the role of BSAP in formation of a non-functional enhanceosome complex and will identify the factors needed for cooperative enhanceosome assembly. Three of the enhancer binding factors (PU.1. Pip, and E47) cooperate to produce powerful transcriptional synergy through PU.1-Pip and E47-Pip DNA binding elements. The mechanism of E47-Pip DNA binding will be approached by footprint, quantitative EMSA, and biosensor (Biocore) studies. We will use computer generated molecular models to guide studies on identifying amino acid residues needed for E47-Pip cooperative DNA binding and transcriptional synergy. We will also determine whether quantitative differences in Pip expression can activate the endogenous kappa gene in pre-B cells. Pip dominant negative mutants will be tested for ability to inhibit endogenous kappa gene expression in plasmacytoma cells. We will also use computer generated molecular models to guide mutagenesis efforts to examine the mechanism of cooperative PU.1-Pip DNA binding and transcriptional synergy. Finally, we will determine the function of specific PU.1 domains in vivo. We previously used biochemical, transient expression, and embryonic stem (ES) cell assays to identify PU.1 domains needed for transcription, protein recruitment to DNA, protein interaction, and macrophage development. We will use homologous recombination technology to prepare knock-in mice that express PU.1 mutants that ablate specific PU.1 functions. The effect of these specific PU.1 mutations on hematopoietic development, transcription, chromatin structure, enhanceosome formation, and somatic recombination will be assessed. These studies represent parallel in vitro and in vivo approaches to study gene regulation and cellular differentiation.